Chenlical Biology Studies On A Novel Lipofuscin Fluorophore ’pdA2E’ And Two Of Important Lipofluscin Pigments In Retinal Pigment Epithelium

Age-related macular degeneration (AMD) is a progerssive chronic disease casused by multiple factors, among which the persistent and chronic light damage to retina is supposed to be the basic cause that lead retinal pigment epithelium (RPE) and photoreceptor to degenarate. As the bisretinoids of lipofuscin are proved to be involed in light damage of retina, it is of great importance to study on bisretiniods.This disseration consists of two parts as follows:1. A novel lipofuscin from human and bovine RPE (retinal pigment epithelium) was confirmed. By 1D- and 2D-NMR (nuclear magnetic resonance)and MS (mass spectrum), its structure was identified and it was named pdA2E (pentadien N-retinylidene-N-retinyl-ethanolamine). It exhibits absorbance maxima (Xmax) at 492 nm and 342 nm, liable to undergo photocatalytic isomerization and oxidation. This fluorophore was also detected in eyecup extracts of Abca4-/- Rdh8-/-(Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargard’s disease model. Excess accumulation of pdA2E in RPE cells was able to significantly affect the cell activity and cause membrance damage. pdA2E was formed when atRAL (all-trans-retinal) reacted with excessive EA (ethanolamine) in the absence of acid, this process may involve the paticipation of three atRAL molecules.2. We investigated the effects of organic solvents which are widely used in processes of biomimetic synthesis and preparation,and extraction and determination of endogenous lipofuscin pigments on two of important lipofuscin, A2E (N-retinylidene-N-retinyl-ethanolamine) and ATR-dimer (all-trans-retinal dimer).1) A2E and ATR-dimer were incubated in DMSO、MeOH、EtOH、THF、chloroform and cyclohexane (only for dimer) and then subjected to high performance liquid chromatography (HPLC). The results showed that A2E and ATR-dimer were both altered by THF and chloroform, but both kept stable in DMSO or MeOH. In addition, ATR-dimer remained unchanged in cyclohexane and EtOH, while A2E was altered in EtOH.2) New processes were designed to extract and quantify lipofuscin in eyecups of mice. Compared with traditional process, the new designed proved higher efficiency.On one hand, this study provides new evidence to the effect of lipofuscin accumulation on RPE cells, indicating a new pathological metabolic pathway of all-trans-retinal in retina. Therefore, this study expands the knowledge of unknown components of lipofuscin and proposes a probable therapeutic target for AMD. On the other hand, we demonstrate the influence of organic solvent on lipofuscin in vitro, and propose effective modification to classical methods. The study further describes the property of lipofuscin components, leading us to a better and more reasonable method to analyze endogenous lipofuscin. Thus this.study has laid a foundation to a more promising investigation on the mechanism of AMD.